The long-term objective of this proposal is to elucidate the basic mechanisms involved in the process of endocytosis. Since virtually all eukoryotic cells use this process for the uptake of macromolecules, it is one of the most important basic mechanisms in a cell. A disruption of this process can have dire effects, as is seen, for example, in the genetic disorder. Familial Hypercholesterolaemia, in which cells cannot take up cholesterol, resulting in severe arteriosclerosis at an early age. In order to properly diagnose and treat endocytosis-related diseases, a thorough understanding of the basic mechanisms of endocytosis is necessary. The information obtained from the experiments proposed here will greatly contribute to this understanding. In this application it is proposed to study the process of endocytosis using a single gene mutant of Drosophila in which endocytosis is reversibly blocked by raising the temperature to 30 degrees C. At 30 degrees C this mutation specifically blocks the step where pits pinch off to form vesicles, but allows the rest of the endocytotic steps to proceed. This results in a depletion of those structures from which membrane is recycled (vesicles and endosomes), and an accumulation of pits on the plasma membrane. Thus, endocytosis can be synchronized to the pit formation stage by exposure to 30 degrees C, and then allowed to proceed in a synchronized wave by lowering the temperature. Since the cytoplasm is essentially cleared of prelysosomal organelles, this allows a superb morphological view of the flow of membrane (and tracer molecules) from one compartment to the next. By observing with an electron microscope at various times after lowering the temperature, it will be possible to unequivocally determine how membrane and solutes are transferred from coated vesicles to endosomes, and from endosomes to lysosomes or Golgi, as well as how and from which compartments recycling occurs. Two cell types, the nephrocyte (specialized for degradation of waste products) and the oocyte (specialized for sequestration of yolk) will be investigated as model systems for the process of endocytosis. In the vitellogenic oocyte, receptor-mediated endocytosis is induced by vitellogenin, a precursor of protein yolk. Thus, endocytosis can be controlled by the presence or absence of vitellogenin, as well as by temperature. Using these two control mechanisms, the role of receptor-ligand binding in (1) pit formation, (2) clathrin binding, and (3) pinch off, will be investigated.